Purification of proteins

ABSTRACT

When proteins are purified using a protein-binding dye immobilized on a chromatographic matrix, the dye or a portion/derivative may leak into the eluant. An ion-exchange resin (e.g. Dowex-1) and a disrupting material (e.g. salt and a fatty acid such as sodium octanoate) are used to separate the dye from the protein to overcome the protein of the leaking dye.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.08/398,484, filed on Nov. 22, 1994, now U.S. Pat. No. 5,625,041, whichis a continuation of application Ser. No. 08/030,255, now abandoned,which is filed under 35 USC 371 as a national phase application ofPCT/GB 91/01556 on Sep. 12, 1991.

The present invention relates to the purification of proteins. In thisspecification, the term "protein" includes naturally-occurring proteins,non-naturally-occurring proteins and other polypeptides which are largeenough to have a ligand binding site, and the term "purification" means"rendering more pure", rather than conferring a given level of purity.

In the separation of proteins from natural sources or, particularly,from the media of fermentations in which a genetically engineered hostcell produces the protein, a protein-containing liquid is often passedthrough a chromatographic column consisting of a protein-bindingcompound bound to a solid support. The protein-binding compound binds toa ligand-binding site on the protein whilst the other material passesthrough the column and the protein is later eluted from the column in apurer form.

However, a small proportion of the protein-binding compound and/or aportion thereof sometimes elutes with the protein and must later beseparated from the protein, particularly if the protein is intended formedical use. There have been prior proposals simply to absorb the dyeonto a column of cross-linked Sephadex (R.T.M., Pharmacia).

Scopes, R. K., in "Protein Purification, Principles and Practice"(Springer Verlag, N.Y., USA, 2nd Edition, pp 141-157), mentioned thattrace amounts of dye in the eluate from dye-containing columns can beremoved on anion exchangers but did not disclose whether it was theprotein or the dye which should bind to the anion exchanger and did notmention the use of a disrupting agent. GB-A-2 053 296 disclosed the useof, amongst other things, a buffer containing sodium chloride and sodiumcaprylate to elute human serum albumin from an affinity medium. However,what those in the art would then have done, whether or not a dyecontamination problem was perceived, was to dialyse away the salt andcaprylate before further treatment. What we have now found is thatcombining the anion exchanger process with the use of a highsalt/caprylate concentration to disrupt the dye-protein binding allowsefficient separation of the dye from the desired protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE 1 illustrates the structural formula of a dye useable to purifyhuman albumin.

Accordingly, one aspect of the present invention provides a process forremoving some or all of a protein-binding compound from an aqueousliquid containing the protein-binding compound and a protein to which itcan bind or is bound, the process comprising the steps of (1) exposingthe liquid to a disrupting material to disrupt binding of the protein tothe protein-binding material, (2) exposing the liquid to an ion exchangeresin to bind the protein-binding material to the resin and (3)separating the resin from the liquid.

Steps (1) and (2) may be simultaneous or may at least overlap such thatthe liquid is still exposed to the disrupting agent at the time that itis exposed to the resin. Step (3) is usually performed by passing theliquid through a column of the resin such that a solution of theprotein, relatively free of the protein-binding material, is obtained.

The process is particularly well suited to removing synthetic textiledye compounds of the sort which have been disclosed in the literaturefor purifying proteins. Many such proteins (probably thousands) can bepurified by the use of such dyes. To pick just one dye, Cibacron Blue3-GA, this can be used to purify kinases, dehydrogenases and most otherenzymes requiring adenyl-containing co-factors, for example NADP⁺ andNAD⁺. Such proteins include alcohol dehydrogenase, adenylate cyclase,adenylate kinase, glucose-6-phosphate dehydrogenase, hexokinase,phosphofructokinase and glyceraldehyde-3-phosphate dehydrogenase.Although the Cibacron Blue 3-GA dye will bind to these classes ofproteins, it is also possible to use the Cibacron Blue 3-GA dye topurify proteins that do not have the dinucleotide binding site. Theseinclude albumin, lipoproteins, blood coagulation factors, interferon andthyroxin binding globulin. These dye compounds are usually anionic, inwhich case an anion-exchanger is most appropriate in the process of theinvention, but some are cationic, in which case a cation-exchanger ismost appropriate. The protein-binding compound is preferably apolysulphonated aromatic compound and is most preferably a triazine dye.Procion Brown MX-5BR, Cibacron Blue 3-GA, (suitable for separating humanserum albumin), Procion Red H-8BN (for carboxypeptidase G2), ProcionYellow MX-AG (for IMP dehydrogenase), Procion Red HE-3B (for lactatedehydrogenase), Procion Green H-4G (for hexokinase), Procion Blue MX-4GD(for malate dehydrogenase), Procion Red H-3B (for 3-hydroxybutyratedehydrogenase) and Procion Blue MX-R (for L-lactate dehydrogenase) areexamples. These and others are summarised in the following table:

    __________________________________________________________________________    Group 1  Group 2  Group 3 Group 4  Group 5    __________________________________________________________________________    P Blue MX-7RX             R Black GF                      P Blue H-EG                              P Black H-EXL                                       P Blue H-ERD    C Blue 2-RA             P Blue MX-R                      P Blue H-EGN                              P Blue H-GR                                       C Blue F-R    R Orange 3R             P Brown MX-GRN                      P Blue H-4R                              P Blue MX-G                                       P Brown H-5R    P Red MX-2B             C Brown 3-GRA                      P Blue MX-3G                              P Blue MX-4GD                                       P Green H-4G    P Rubine H-BN             P Navy H-4R                      C Blue F3-GA                              D Blue K-BL                                       P Green H-E4BD    P Turquoise H-A             P Orange MX-G                      R Blue B                              P Brown H-3R                                       P Navy H-ER    P Turquoise MX-G             R Orange FR                      R Blue R                              P Brown MX-5BR                                       P Red H-3B    C Turquoise 6-GE             P Red MX-5B                      C Navy F-2R                              P Orange H-ER                                       P Red H-8BN    R Violet R             P Scarlet MX-G                      P Red H-E3B                              P Orange MX-2R                                       P Red H-E7B    R Yellow GNL             P Scarlet MX-3G                      P Rubine MX-B                              P Red MX-7B                                       P Scarlet H-E3G    P Yellow H-A             C Turquoise GFP                      P Scarlet H-2G                              P Red MX-8B                                       P Yellow H-E3G    P Yellow MX-6G             C Yellow R-A                      P Yellow H-E6R                              C Red 3-BA                                       P Yellow H-E6G    P Yellow MX-8G             P Yellow MX-3G                      P Yellow H-5G                              P Violet H-3R                                       P Yellow H-E4R             P Yellow MX-4R                      P Yellow MX-R                              P Yellow H-E6R                                       P Yellow MX-GR                      C Yellow 3-GP    __________________________________________________________________________     Group 1 dyes bind the least protein from crude extracts of tissues, and     group 5 dyes the most. Actual groups may vary ± 1 with different types     of extract. P, I.C.I. Procion; C, CibaGeigy Cibacron; R, Hoechst Remazol;     D, Sandoz Drimarene. Not all of these dyes are still commercially     available. Source: From J. Chromatogr. 376, 131-140 (1986)

The dye itself (with or without the spacer which is commonly used toattach the dye to a column) may cause the contamination, or the problemmay be caused by a derivative of the dye or an intermediate used in thesynthesis of the dye.

Cation-exchangers include S and CM Fast Flow, from Pharmacia.

Anion-exchangers include Pharmacia's DEAE Fast Flow and Q Fast Flow.Preferably, the matrix is Dowex-1, which is a strongly basic anionexchange resin, preferably 2% cross-linked, with a dry mesh size of50-100. Generally, a strong anion exchanger is better than a weakexchanger.

The protein may be a serum-derived protein such as human albumin, alipoprotein, a blood coagulation factor such as Factor VIII or FactorIX, thyroxin-binding globulin or alpha interferon. Preferably, theprotein is human albumin (HA) or a mutant or fragment thereof whichretains a dye-binding domain (such as is described in EP-A-322094published Jun. 28, 1989) or a fusion of HA or a said mutant orfragment with another protein. The aqueous liquid is suitably the director indirect result of exposing a fermentation medium or fractionsthereof to the protein-binding compound; "indirect" in this contextmeans that the fermentation medium, after contact with theprotein-binding compound, may be treated in one or more process stepsbefore the process of the invention is applied. By "fermentation medium"we mean the medium which results from the fermentation of an organismcapable of producing the protein. The organism (which term includes celllines) is preferably transformed or transfected to produce the proteinand the protein is normally heterologous to the. organism. The organismmay be a bacterium (eg E. coli or B. subtilis), a yeast (egSaccharomyces cerevisiae), a non-yeast fungus (eg Aspergillus tiger) ,an insect cell (eg Spodoptera frugiperda), a plant cell (eg a hairy rootcell culture of Atropa belladonna) or a mammalian cell (eg Vero cells).Preferably, the organism is a yeast. Exemplary genera of yeastcontemplated to be useful in the practice of the present invention arePichia, Saccharomyces, Kluyveromyces., Candida, Torulopsis, Hansenula,Schizosaccharomyces, Citeromyces, Pachysolen, Debaromyces,Metschunikowia, Rhodosporidium, Leucosporidium, Botryoascus,Sporidiobolus, Endomycopsis, and the like. Preferred genera are thoseselected from the group consisting of Pichia, Saccharomyces,Schizosaccharomyces, Kluyveromyces, Yarrowia and Hansenula, because theability to manipulate the DNA of these yeasts has, at present, been morehighly developed than for the other genera mentioned above.

Examples of Saccharomyces are Saccharomyces cerevisiae (especiallypreferred), Saccharomyces italicus and Saccharomyces rouxii. Examples ofkluyveromyces are Kluyveromyces fraqilis and Kluyveromyces lactis.Examples of Hansenula are Hansenula polymorpha, Hansenula anomala andHansenula capsulata. Yarrowia lipolytica is an example of a suitableYarrowia species, and Schizosaccharomyces pombe is a further suitableyeast.

The production of human albumin expressed from a gene inserted into asuitable host by recombinant DNA techniques is well known in the art anddoes not require discussion herein. Examples of specific prior artprocesses include those described in EP-A-147 198 (Delta Biotechnology),EP-A-201 239 (Delta), EP-A-60 057 (Genentech), EP-A-88 632 (Genentech),EP-A-251 744 (Delta) and EP-A-286 424 (Delta).

Similarly, processes for purifying proteins from a fermentation mediumare known in the art. A good review may be found in "ProteinPurification--Principles and Practice", 2nd Edition (Springer Verlag,N.Y.), especially pages 141-157.

Preferably, the aqueous liquid results from passing the fermentationmedium through one or more separation (eg chromatographic) steps.

It is to be noted that, although the process of the invention isparticularly well suited to separating a protein-binding compound from aprotein when the protein-binding compound has been used to purify theprotein from, for example, a fermentation medium or a product thereof,the process can generally be used to separate any suitableprotein-binding contaminant from a protein. An advantage of the processis that it does not require binding o. the protein to the resin andhence relatively large volumes of protein can be purified for a givenvolume of resin.

The disrupting material may be a single compound or a mixture.Preferably, it comprises a mixture of a salt (preferably sodium chlorideor potassium chloride) and a compound to disrupt hydrophobicinteractions between the protein and the protein-binding compound, forexample a (preferably non-ionic) detergent, an organic so sent or,preferably, a fatty acid. Alternative disrupters of hydrophobicinteractions with the protein include N-acetyltryptophan and mandelicacid, which will normally be used as their salts, for example sodiumsalts. The fatty acid is preferably octanoic acid but other fatty acids(preferably C₆ -C₁₀ and preferably saturated) may be used. The fattyacid will usually be present in the form of its salt, for example thesodium salt. The concentrates of the salt and fatty acid may be variedto suit the particular protein and protein-binding compound in question.A salt concentration of 0.1 M to 3 M will generally be useful,preferably 0.5 to 2.0 M. A fatty acid concentration of 10 mM-100 mM isgenerally useful, preferably 25-60 MM, most preferably about 50 mM. Whenthe disrupting material is a single compound, any of these materials maybe used.

The liquid which is exposed to the ion exchange resin will usuallyconsist largely of the buffer used to elute the protein from the columncontaining the protein-binding compound. The disrupting material or acomponent of it may then be added. For example, if the elution buffercontains 2 M NaCl in a 50 mM phosphate buffer of pH 7.0, there may be noneed to add further salt, and only the fatty acid is added. The pH canbe altered if desired. We have found that a pH of about 7.0 is suitable,but generally any pH of above 5.0 is applicable to any fatty acid.

The pH should preferably be such that the protein-binding compound ischarged; for example most polysulphonated triazine dyes are negativelycharged above pH 2 to 3. It is not always necessary for the liquid tocontain a buffer.

The most convenient means of exposing the mixture of the protein andprotein-binding compound to the ion exchange resin and disruptingmaterial will be to add the disrupting material to the mixture and thento pass the resulting liquid through a column of the ion exchange resin.This minimises the amounts of buffer and resin used, and the amount ofprotein lost. However, it is technically possible to expose theprotein/protein binding compound mixture to the resin first, and then toelute the protein with a buffer containing the disrupting material. Alarger column of resin will usually be needed in such an embodiment,which will then probably have to be cleaned stringently with suitableacids and solvents rather than being simply discarded.

The columns may be the conventional linear type or radial flowcartridges.

The invention will now be illustrated by way of example and withreference to FIGURE 1 which shows the structure of a textile dye(Cibacron Blue 3-GA) and spacer (4-amino butyl group usable in a columnto purify human albumin.

EXAMPLE 1

As a model of the product of passing an HA-containing fermentationmedium through a purification column, a 3 mg.ml⁻¹ solution of humanserum albumin was prepared in 2 M NaCl, 50 mM phosphate buffer pH 7.0,and 21 μg.ml⁻¹ of Cibacron Blue dye covalently attached to a spacer(FIGURE 1) was added. The dye included a spacer used to attach the dyemolecule to the matrix and also a dye synthesis intermediate. 1 M sodiumoctanoate, as the disrupter of hydrophobic interactions, was added togive a concentration of 50 mM. This solution (20 ml) was then passedthrough a 1 ml column of Dowex-1 resin (2% cross-linked; Dow ChemicalCo) at a flow rate of 0.5 ml.min⁻¹. The removal of blue dye from HA wasmeasured spectrophotometrically at 620 nm.

Under these conditions, about 97% of the blue dye bound to the resin.The unbound fraction which had passed through the column containedgreater than 97% of the HA applied to the column.

EXAMPLE 2

Following the procedure of Example 1, the efficiency of dye removal fromHA was assessed in the presence of buffer, 2 M NaCl, caprylate andcombinations of these components. As can be seen from the results inTable 1, a combination of salt and fatty acid was much more effectivethan the individual components.

                  TABLE 1    ______________________________________                    Dye + Spacer                               Dye Intermediate                    Removal    Removal    Buffer          (%)        (%)    ______________________________________    A    50 mM phosphate pH 7.0                        19         N/D    B    50 mM phosphate +                        32         N/D         2 M NaCl    C    50 mM phosphate +                        46         N/D         50 MM caprylate    D    Combination (B + C)                        97         96    ______________________________________     N/D = not determined

EXAMPLE 3

The comparison of Example 2 was repeated, using Cibacron Blue 3-GA(Blue), Procion Green H-4G (Green), Procion Brown MX-5BR (Brown) andProcion Red HE-3B (Red) dyes covalently attached to a spacer. Theresults are shown in Table 2.

                  TABLE 2    ______________________________________            HSA/Dye + Spacer Separation (% removal)    Buffer    Blue   Green        Brown Red    ______________________________________    A         19     11           52    33    B         32     41           93    57    C         46     45           90    83    D         97     65           89    92    ______________________________________     A-D as Table 1

EXAMPLE 4

The experiment of Example 2 was repeated with different proteins. Theresults are shown in Table 3. Alkaline phosphatase was mixed with blueor red dyes.

                  TABLE 3    ______________________________________    Protein/Dye + Soacer Separation (% removal)    Buffer          HSA    LACTOFERRIN ADH  GK   AP/Blue                                              AP/Red    ______________________________________    A     19     66          N/D  N/D  52     N/D    B     32     N/D         42   73   80     63    C     46     67          72   96   80     71    D     97     81          94   91   84     92    ______________________________________     A-D as Table 1     GK = glycerokinase     ADH = alcohol dehydrogenase     AP = alkaline phosphatase     N/D = not determined

We claim:
 1. In a process for purifying a protein, said processcomprising:(a) providing a chromatographic column comprising a solidsupport and bound thereto a protein-binding compound which willreleasably bind the protein; (b) passing a solution of the proteinthrough the column such that the protein-binding compound releasablybinds the protein; and (c) eluting the protein from the column to yielda liquid containing protein and any protein-binding compound in boundform that eludes; wherein the improvement comprises the further stepsof:(d) exposing the liquid to an ion exchange resin under conditionssuch that the ion exchange resin does not directly bind the protein, butdoes bind the protein-binding compound; and (e) separating the resinhaving the protein-binding compound bound thereto from the liquidcontaining said protein, wherein, prior to either step (d) or step (e),the liquid is exposed to a disrupting material to disrupt binding ofsaid protein to said protein-binding compound, said disrupting materialcomprising a mixture of a salt and a compound, other than saidprotein-binding compound, which disrupts hydrophobic interactionsbetween the protein and said protein-binding compound.
 2. A processaccording to claim 1, wherein said steps (d) and (e) are carried outsimultaneously by passing said aqueous liquid through a columncontaining said resin and eluting said column with a suitable liquid. 3.A process in accordance with claim 1, wherein said disrupting materialis admixed with said aqueous liquid prior to passage through saidcolumn.
 4. A process in accordance with claim 1, where said disruptingmaterial is admixed with said liquid utilized to elute said column.
 5. Aprocess in accordance with claim 1, wherein the compound which disruptshydrophobic interactions between the protein and said protein-bindingcompound is a fatty acid or a salt thereof.